Research paper

Attenuation of Wnt/β-catenin signaling in patients with Stevens-Johnson

syndrome and toxic epidermal necrolysis

Chun-Bing Chen1,2,3,4,5,6, Wan-Chun Chang1, Ming-Ying Wu1,4, Tzu-Yang Kao1, Ying-Wen Wang1,

Chuang Wei Wang1,2,3, Chi-Ju Chen7, Wen-Hung Chung1,2,3,4,6,8 , Shih-Chi Su1,2

1 Department of Dermatology, Drug Hypersensitivity Clinical and Research Center, Chang

Gung Memorial Hospitals, Linkou, Taipei, and Keelung, Taiwan

2 Whole-Genome Research Core Laboratory of Human Diseases, Chang Gung Memorial

Hospital, Keelung, Taiwan

3 Chang Gung Immunology Consortium, Chang Gung Memorial Hospital and Chang Gung

University, Taoyuan, Taiwan

4 College of Medicine, Chang Gung University, Taoyuan, Taiwan

5 Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University,

Taoyuan, Taiwan

6 Immune-Oncology Center of Excellence, Chang Gung Memorial Hospital, Linkou, Taiwan

7 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan

8 Department of Dermatology, Xiamen Chang Gung Hospitals, China

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Correspondence to:

Wen-Hung Chung, MD, PhD

Director, Drug Hypersensitivity Clinical and Research Center,

Department of Dermatology, Chang Gung Memorial Hospital

Professor, School of Medicine, Chang Gung University

5 Fusing St. Taoyuan, 33305, Taiwan

Telephone: +886-3-3281200-2216

Fax: +886-2-27191623

E-mail: wenhungchung@yahoo.com; chung1@cgmh.org.tw

Dr. Shih-Chi Su, PhD

Whole-Genome Research Core Laboratory of Human Diseases, Chang Gung Memorial

Hospital, Keelung, 204, Taiwan

Tel: 886-2-27135211#3397; Fax: 886-2-27191623; E-mail: ssu1@cgmh.org.tw

Abstract: 200 words

Manuscript: 3929 words

Numbers of figures: 6

Numbers of tables: 1

Number of references: 48

Supplement: 1

Short title: Wnt/β-catenin signaling in SJS/TEN

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Abstract：Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN) are rare but life-

threatening severe cutaneous adverse reactions. Current studies have suggested that the

pathobiology of drug-mediated SJS/TEN involves a dysregulation of cellular immunity with

overwhelming activation of cytotoxic T lymphocytes. The canonical Wnt signaling pathway

plays important roles in T cell development and activation, which may provide potential

avenues for alleviating dysregulated immunity in SJS/TEN. In this study, we aimed to assess

the implication of Wnt signaling in drug-reactive T cells in SJS/TEN. We showed

downregulation of Wnt signaling components, including T cell factor 1 (TCF-1)/lymphoid

enhancer binding factor 1 (LEF-1) transcription factors, in SJS/TEN patients, suggesting that

canonical Wnt signaling is regulated during cytotoxic T cell responses in SJS/TEN. Further

analyses demonstrated that engagement of the T cell receptor by antigen encounter and

treatment of a prognostic marker of SJS/TEN, IL-15, in vitro led to the downregulation of

LEF-1 and TCF-1 expression in CD8+ T cells. Enhancement of Wnt signaling by adding the

Wnt activators attenuated ex vivo activation of drug-specific T cells from SJS/TEN patients,

indicating a functional involvement of Wnt signaling in the pathomechanism of SJS/TEN.

These findings provide additional insight into the immunopathogenesis and therapeutic

intervention of this devastating condition.

Keywords：Stevens-Johnson syndrome (SJS), toxic epidermal necrosis (TEN), cytotoxic T lymphocyte

(CTL), Wnt, T cell factor-1 (TCF-1), lymphoid enhancer binding factor 1 (LEF-1)

Short Title:

Wnt/β-catenin signaling in SJS/TEN

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Abbreviations: SJS, Stevens-Johnson syndrome

TEN, toxic epidermal necrolysis

SCORTEN, severity of illness score of toxic epidermal necrolysis

RegiSCAR, Registry of Severe Cutaneous Adverse Reactions

BSA, body surface area

CTL, cytotoxic T lymphocyte

NK, natural killer

MHC, major histocompatibility complex

PBMC, peripheral blood mononuclear cell

LEF-1, lymphoid enhancer binding factor 1

LTT, lymphocyte transformation test

MPE, maculopapular exanthema

DRESS, drug reaction with eosinophilia and systemic symptoms

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Introduction:

Stevens-Johnson syndrome(SJS) and toxic epidermal necrolysis(TEN) are considered a

spectrum of life-threatening severe cutaneous adverse reactions. SJS/TEN share similar

clinical presentations like rapidly progressing blistering exanthema and target-like macules.

The affected skin specimens typically showed subepidermal blistering, extensive apoptotic

keratinocytes and partial- or full-thickness epidermal necrosis and epidermal detachment

with a sparse dermal T-cell lymphocyte infiltrates histopathologically.[1, 2] Mucosal

involvements and skin detachments are also characteristic.[3] SJS is defined by the degree of

skin detachment less than 10% of body surface area(BSA), while TEN is defined as the area

of denuded skin greater than 30% of BSA. Besides, the cases with degree in-between(10–

30%) are diagnosed as SJS/TEN overlap.[4] Though the incidence is relative low[5, 6], they

may cause multi-organ failure and significant mortality, with the mortality rate being 10% for

patients with SJS, approximately 30% for patients with SJS/TEN overlap and almost 50% for

patients with TEN.[7, 8]

Although the underlying pathological mechanisms are not fully understood, current

pharmacogenomic studies have proposed an association between drug-induced SJS/TEN and

human leukocyte antigen(HLA) genes.[9-11] Specific HLA molecules may have higher binding

affinities for drug antigens and present the drug antigens to T cell receptor(TCR), causing a

cascade of T cell activations and aberrant immune responses directed at keratinocytes.[3,

12, 13] Though the discovery of the predisposing gene has lowered the incidence, present

therapies are mostly empirical.[14] The situation point out the fact that there are numerous

unanswered questions remain with regard to the immunological and cytotoxic signaling

pathways.

The Wnt signaling pathway is evolutionarily conserved and is implicated in a large

variety of developmental processes including specification of cell fate and maintenance of

stem cell pluripotency.[15, 16] In parallel, aberrant Wnt signaling underlies a wide range of

pathophysiological conditions.[17] This pathway utilizes T cell factor(TCF)/ lymphoid

enhancer binding factor(LEF) transcription factors and β-catenin coactivator to achieve

balanced regulation of its downstream gene expression. It is well established that several

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Wnt ligands and their effector proteins are crucial for normal T cell development in thymus.

[18] Recent studies have also revealed critical requirements for Wnt signal transduction

cascade in regulation of mature T cell responses in the circulation.[19] Specifically, Wnt

pathway orchestrates the generation and persistence of functional memory CD8+ T cells,

promotes Th2 differentiation, and suppresses Th17-differentiation of activated CD4+ T cells.

[20] Activation of β-catenin facilitated CD8+ memory T cell formation, with enhanced

protective capacity and extended survival of CD4+CD25+ regulatory T cells(Tregs).[21]

The cytotoxic CD8+ T cells, that are vital in defense against pathogens and non-self

antigens(e.g. medication in adverse drug reactions), remain the key player in the

pathomechanism of SJS/TEN. Activation of drug-specific naive CD8+ T cells requires TCR

stimulation, costimulation, and proinflammatory cytokines from antigen-presenting cells

and other innate immune cells.[12] Activated T cells then undergo massive clonal expansion

and are equipped with cytokines such as interferon-γ and cytolytic molecules, including

granzyme B, perforin, and granulysin.[14] Yet, only a small portion of activated cells further

differentiates into memory CD8+ T cells, which subsequently contribute to the devastating

condition in SJS/TEN, a delayed-type hypersensitivity reaction while re-exposed to the same

culprit drug. A role of Wnt signaling pathway in promoting generation of memory CD8+ T

cells has been demonstrated based on the observation that antigen-primed human memory

CD8+ T cells expressed lower TCF-1 and LEF-1, two downstream effectors of Wnt signal, than

did naive T cells.[22] Consistently, constitutive activation of the Wnt signaling pathway was

found to reduce effector CD8+ T cell expansion.[23, 24] Another recent study confirmed

these findings and further demonstrated that forced expression of stabilized β-catenin in

naive T cells interfered with proximal TCR signaling.[25] These data suggest that modulating

the activity of Wnt pathway may be a potential niche to mitigate dysregulated immunity and

treat T cell-mediated diseases, such as SJS/TEN. However, little is known about the role of

Wnt signaling pathway in the pathobiology of SJS/TEN. In this study, we assessed the

implication of Wnt signaling in drug-reactive T cells and also determined whether the Wnt

signaling pathway is functionally associated with the disease activity in SJS/TEN.

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Method:

Subject recruitment and sample collection

Patients who fulfilled the consensus diagnostic criteria for SJS/TEN were enrolled at the

Chang Gung Memorial Hospital Health System. All cases were evaluated by at least two

dermatologists, who reviewed all available photographs, histological data, and clinical

information, including type of cutaneous reactions, date of onset, and drug history, dosage,

and duration. Phenotypes were clinically assessed using the diagnostic criteria established.

[26] Plasma or serum samples, and PBMC of patients were obtained from whole blood

samples as well as blister cells within 2 days after admission(1-7 days after the index day) at

the active stage of disease course(before systemic immunomodulatory or

immunosuppressive treatment) and after complete skin reepithelization at recovered stage.

Blister fluid was collected by puncture of several blisters at the active stage while applicable.

In addition to SJS/TEN, blood samples from burn injury patients (n=11) and heathy donors

(n=11), and blister fluid from burn injury patients (n=11) were used as control groups.

Informed consent was obtained from the participants.

Quantitative RT-PCR analysis

Total RNA was extracted from cells indicated by TRIzol reagent (Invitrogen, Carlsbad,

CA) and checked for quality by the Bioanalyzer 2100 system (Agilent Technologies, CA). An

AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Stratagene) was used to prepare

cDNA. Primers were designed using Beacon Designer software. Reactions were analyzed on

a Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection system using iQ SYBR Green

Supermix (Bio-Rad, Hercules, CA). Each real-time PCR reaction contained 0.5 ng/ul of cDNA

and 400 nM of each primer in a 25-ul reaction volume. The reaction was initiated at 94°C for

1.5 min, followed by 40 two-step amplification cycles consisting of 15 s of denaturation at

95°C and 45 s of annealing/elongation at 60°C. A final dissociation stage generated a melting

curve for verification of amplicon specificity. Assays were performed in triplicate.

FACS staining

Cells were subject to methanol permeabilization before examining intracellular

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expression of LEF1 and TCF-1 within CD8+ T cells. LEF1 and TCF-1 were detected by indirect

staining using pretitrated mAbs REMB6 (Oncogene Research Products) and 7H3 (Upstate

Biotechnology), respectively. Briefly, 2 ug of primary mAb were added to 106 cells and

incubated for 1 h at room temperature. After washing, this is followed by staining with

corresponding 2nd Ab (DakoCytomation) for 1 h at room temperature. Cells were

subsequently washed in blocking buffer before surface staining with directly conjugated

mAbs specific for CD56 or CD8 (BD Biosciences). T he fraction of TCF1 high and LEF1 high in CD8 T

cell subsets were further analyzed and compared between the active stage and the recovery

stage in 4 SJS/TEN patients. Furthermore, to illustrate the restriction of stimulation by

specific offending drugs, we compared the expression of LEF1 and TCF-1 within CD8+ T cells

among groups stimulated by culprit drugs (including 5 SJS/TEN cases: 2 carbamazepine, 1

lam o trigine, 1 oxcarbazepine, and 1 moxifloxacin-induced SJS/TEN cases) and tolerant drugs

(including phenytoin, clonazepam, and cefazolin).

Lymphocyte activation test (LAT)

PBMCs (106/ml), obtained from the heparinized blood by density centrifugation on

Ficoll-Hypaque, were cultured in RPMI1640 medium supplemented with heat-inactivated

AB-serum, glutamine, antibiotic–antimycotic solution and nonessential amino acids. All

cultures were performed in triplicate in 96-well plates. Stimuli are the culprit drugs

(including 1 carbamazepine and 1 ketoprofen-induced SJS/TEN cases) and the pan T-cell

mitogen phytohaemagglutinin (PHA) as a positive control. After 7 days, the stimulation index

(SI) was calculated by the level of secreted granulysin of stimulated to unstimulated

cultures. The levels of granulysin were determined by home-made ELISA as described

previously, [27] whose sensitivity for granulysin is 2.5 ng/mL.

Evaluation of the circulating Wnt agonists and antagonists

Concentrations of DKK1, SOST, WIF1, and Wnt3a in sera or blister fluid were

determined by commercial ELISA(R&D systems, Minneapolis, Minnesota). All samples will be

analyzed in triplicate.

Statistical analyses

Differences in the levels of biomarkers or cell responses between groups were

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evaluated by the Student’s t-test. The paired-sample t-test was applied in the experiments

where samples are analyzed in different time points. A p value<0.05 was considered

significant. The data were processed by using SAS statistical software(Version 9.1,2005; SAS

Institute Inc., Cary, NC).

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Result:

Study subjects

In this study, serum samples from total 25 SJS/TEN patients were used to explore the

potential role of Wnt pathway in SJS/TEN. All the cases were collected at the Chang Gung

Memorial Hospital Health System which received referral patients from the Taiwan Severe

Cutaneous Adverse Reaction Consortium across Taiwan. The distribution of demographic

characteristics, phenotypes, and underlying conditions, and causative drugs among patients

and controls are shown in Table 1 and Supplement Table 1 , respectively . The mean age of

the study group is 52.5±18.7 years. There are 7 cases present with SJS, 6 with SJS/TEN

overlap, and 12 with TEN. In average, these patients have erythema covered more than half

of the total body surface area while blisters or detachments involved 30% of the total body

surface area (56.5 26.7 and 30.9 22.9, respectively). The mortality rate is 20.0%. For

mucosal involvement, the results show a hundred percent of oral ulcers clinically, followed

by genital ulcers and ocular involvements. The most common complications are hepatitis

and gastrointestinal bleeding (n=6, 24.0%). The presence of atypical lymphocytes in blood

draw was observed in 64.0% of the patients, and 24.0% of the patients have eosinophilia.

16.0% and 36.0% of the patients have leukocytosis and leukopenia respectively while suffer

from hypersensitivity reactions. In addition, 28.0% of the patients have thrombocytopenia.

Fever episode is also a common presentation and affects more than half of the study

group(n=13, 42.0%). Three patients suffered from permanent corneal damage and visual

impairments. As for the culprit drugs that these patients used, the offending drugs are

mainly anticonvulsant agents(n=8), antibiotics(n=7) and allopurinol(n=4).

Aberrant expression of Wnt signaling components in PBMCs and blister cells from SJS/TEN

patients as well as drug-activated T cells from SJS/TEN patients

Mounting evidence has shown that Wnt signaling pathway is involved in the regulation

of mature T cells.[22, 28-30] Upon the activation of effector T cells, components of the Wnt

signaling pathway, such as active form of β-catenin(dephosphorylated state), LEF1, TCF1,

lipoprotein receptor-related protein-5(LRP5, Wnt coreceptor), LRP6(Wnt coreceptor), are

differentially expressed. To determine the putative fluctuations in Wnt signaling during the

progression of SJS/TEN, expression levels of genes related to Wnt signaling in T cell function,

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including LEF1, TCF1, LRP5, and LRP6, were assessed in blister cells from SJS/TEN patients

and compared with that in PBMCs from SJS/TEN patients and from normal subjects. We

showed that a decrease in gene expression for LEF1, TCF1, and LRP6 was detected in

SJS/TEN as compared with normal subjects(Figure 1A). Further, we examined the expression

of these genes in drug-activated T lymphocytes from SJS/TEN patients. Similar results were

obtained in T lymphocytes from SJS patients which were exposed to the causative

drug(Figure 1B). However, such downregulation was not observed for LRP5 in PBMCs,

blister cells, or drug-activated T lymphocytes from SJS/TEN.

TCF1 and LEF1 protein is downregulated in CD8+ T cells at the active stage of SJS/TEN.

To further determine whether Wnt signaling is fluctuated in CD8 T lymphocytes during

the progression of SJS/TEN, we also examined LEF1 and TCF-1 protein expression in

peripheral CD8 T cell subsets by intracellular FACS staining. As shown in Figure 2, a decrease

in the protein levels of both TCF1 and LEF1 was detected in the CD8 T cells of SJS/TEN

patients at the active stage of disease course as compared with that at recovered stage. The

portion of TCF1high and LEF1high CD8 T cells at the active stage was smaller than that at the

recovered stage, suggesting that Wnt signaling is repressed in the key effector cells involved

in SJS/TEN.

Endogenous Wnt inhibitors, DKK1 and WIF1, are increased in SJS/TEN

Given the sufficiency of LEF1/TCF1 in modulating T cell responses during drug

hypersensitivity reaction, a critical question is how this pathway is regulated in SJS/TEN.

Many secreted proteins are known to serve as endogenous regulators of Wnt signaling(e.g.

DKK1, SOST, WIF1, and Wnt3a). We, thus, tested whether these endogenous regulators of

Wnt signaling are regulated in SJS/TEN by measuring the levels of DKK1, SOST, WIF1, and

Wnt3a in the sera from SJS/TEN patients and normal subjects. We found that two

endogenous Wnt inhibitors, DKK1 and WIF1, were elevated in the sera of SJS/TEN compared

to health donors(Figure 3A). Furthermore, the levels of these endogenous Wnt regulators in

the blister fluid from the SJS/TEN and burn patients were also evaluated. The levels of DKK1

and WIF1 in blister bluid of SJS/TEN patients were higher than that in blister fluid of burn

patients(Figure 3B). These findings in part provide clues for regulation of Wnt signaling in

SJS/TEN.

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IL-15 contributes to the attenuation of Wnt signaling in CD8+ T cells

The crosstalk between proinflammatory cytokines(e.g. IL-6, IL-8, IL-15, and TNF-a) and

Wnt signaling pathway had been demonstrated.[31, 32] We have recently shown that serum

levels of several cytokines and cytolytic proteins were elevated in SJS/TEN.[33] Of note,

among these cytokines regulated in SJS/TEN, IL-15 was shown to be associated with the

clinical severity and death of this condition.[33] Here, we demonstrated that a decrease in

the portion of TCF1high CD8 T cells was observed as PBMCs from normal subjects were

treated with IL-15 for 24 hours but not with IL-6, IL-8, or SDF-1(stromal cell-derived factor-1,

used as a non-SJS-related cytokine) (Figure 4), revealing an impact of IL-15 on altering the

activation of Wnt signaling in primary CD8+ T cells.

Drug-mediated TCR activation attenuates the Wnt signal transduction

Specific HLA types is highly associated with specific drug induced-SJS/TEN, which

implies that specific HLA molecules may have higher binding affinity for specific drug

antigens and present the drug antigens to specific TCR, further causing T cell activation and

adverse responses.[13] Thus, we further test whether drug-mediated engagement of TCR

affects Wnt signaling in SJS/TEN. For example, PBMCs of a carbamazepine(CBZ)-induced SJS

patient were stimulated with the culprit drug (CBZ) or a non-causative drug (phenytoin, PHT)

for 7 days. The expression levels of genes related to Wnt signaling involved in T cell function

were then examined in CD8+ T cells. We observed that the treatment of CBZ but not PHT

attenuated the expression levels of LEF1 and TCF-1 proteins in CD8+ T cells of CBZ-induced

SJS patients(Figure 5), suggesting that specific TCR engagement may dampen Wnt signaling

in SJS/TEN.

Wnt signaling is functionally implicated in the pathobiology of SJS/TEN

Previous studies have revealed critical requirements for Wnt signaling in regulation of

mature T cell responses.[19] The effect of Wnt signaling on drug-specific activation of T cells

in SJS/TEN, therefore, was examined. To manipulate the Wnt signal pathway, LiCl and SB-

216763, two GSK3 inhibitors, were used to pretreat the drug-specific T cell clones to activate

the Wnt signaling in effector cells. Consistently, the presence of LiCl or SB-216763 repressed

the activation of drug-specific T cell in SJS/TEN (Figure 6A and 6B), indicating a functional

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involvement of this signal pathway in the pathomechanism of SJS/TEN. 1

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Discussion:

CD8+ T cells and TCR function play a key role in the development of SJS/TEN, which is

characterized as widespread epidermal and mucosal necrosis.[34] Cytotoxic CD8 T cells that

produce cytotoxic molecules (such as granulysin and granzyme B) to cause extensive

keratinocyte death, are enriched in blister fluid samples from the skin lesions of patients

with SJS/TEN.[27, 35] These cytotoxic cells mediate the disease pathogenesis and are

correlated with the disease severity and mortality. In this study, we showed that Wnt

signaling components, including LEF1, TCF1, and LRP6, were downregulated in SJS/TEN

patients, while the serum levels of endogenous Wnt inhibitors, DKK1 and WIF1, were

elevated. These findings revealed that canonical Wnt signaling is regulated in SJS/TEN.

Further assays demonstrated that engagement of the TCR by the specific causative drug and

the treatment of a biomarker of SJS/TEN, IL-15 in vitro led to the downregulation of LEF-1

and TCF-1 expression in CD8+ T cells from SJS/TEN patients. Manipulation of Wnt signaling

by adding the Wnt activators attenuated ex vivo activation of drug-specific T cells from

SJS/TEN patients. Our data here, for the first time, suggest a functional involvement of Wnt

signaling in the pathomechanism of SJS/TEN.

The canonical Wnt signaling transduction cascade is an evolutionarily conserved and

multi-functional pathway and plays a crucial role in T cell development, differentiation, and

functionality.[36] Upon the activation of the Wnt signal pathway, naive CD8+ T cells undergo

clonal expansion and further differentiate into effector and memory precursors. The Wnt

signal pathway negatively regulates differentiated effector CD8+ T cells and positively

upregulates memory precursor CD8+ T cells through TCF-1 alone or its combination with β-

catenin.[19] In addition, a role of Wnt signaling pathway in promoting generation of

memory CD8+ T cells has been demonstrated through the observation that antigen-primed

human memory CD8+ T cells expressed lower TCF-1 and LEF-1 than did naive T cells. [22]

Consistently, constitutive activation of the Wnt signaling pathway was found to reduce

effector CD8+ T cell expansion.[23, 24] Another study also confirmed these findings and

further demonstrated that forced expression of stabilized β-catenin in naive T cells

interfered with proximal TCR signaling.[25] It is now clear that Wnt/β-catenin pathway is a

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key signaling pathway governing CD8+ T cell differentiation. However, little is known about

the role of Wnt signaling pathway during the course of SJS/TEN.

The Wnt signaling pathway utilizes TCF-1/ LEF-1 transcription factors and β-catenin

coactivator to achieve balanced regulation of its downstream gene expression upon the

engagement of Wnt and its various receptors. Upon the activation of effector T cells,

components of the Wnt signaling pathway, such as active form of β-catenin

(dephosphorylated state), LEF1, TCF1, lipoprotein receptor-related protein-5 (LRP5, Wnt

coreceptor), LRP6 (Wnt coreceptor), matrix metalloproteinase 2 (MMP2, Wnt target gene),

and MMP9 (Wnt target gene) are differentially expressed.[29, 37] In this study, we found

that genes encoding Wnt signal components, including LEF1, TCF1, and LRP6, were

downregulated in PBMC as well as in blistering cells from SJS/TEN as compared with normal

subjects. Our study further demonstrated decreased intracellular LEF1 and TCF1 protein

expression in CD8 T cell subsets of SJS/TEN patients, suggesting attenuation of Wnt signaling

in CD8 T cells in SJS/TEN. We have previously shown that a number of cytokines was

upregulated in SJS/TEN patients, of which IL-15 was significantly correlated with the disease

severity and mortality of SJS/TEN.[33] Here, we found that Wnt singals was attenuated

specifically by IL-15 but not IL-6 or IL-8. In addition to the impact from SJS-related cytokines,

Wnt signaling was dampened in CD8 T cells from SJS patients by TCR engagement with the

culprit drug antigen. Moreover, we detected that endogenous Wnt inhibitors, DKK1 and

WIF1, were elevated in the sera and bliser fluid of SJS/TEN compared to that from healthy

donors and burn patients. These findings collectively provide clues for how Wnt signal

pathway is regulated in the CD8 T cells in SJS/TEN. Furthermore, the presence of Wnt

activators attenuated ex vivo activation of drug-specific T cells from SJS/TEN patients,

indicating a functional involvement of Wnt signaling in the pathomechanism of SJS/TEN and

a reasonable therapeutic implication.

In this study, we found that the LRP5 was not simultaneously downregulated compared

with LRP6 in SJS/TEN though no statistical significance noticed. Active Wnt signaling is

initiated by binding of a Wnt protein to its receptor (Frizzeld) and one of the coreceptors

(LRP5 or LRP6). LRP5 is similar in sequence and structure to LRP6, and these two receptors

have been proposed to function largely in the same contexts and signaling pathways, but

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there are difference between the two receptors. LRP5 usually can act as a gatekeeper for

Wnt responses to enable Wnt pathway responsiveness. [ 38 ] Comparing with LRP6, which

exhibits strong signaling activity, LRP5 was reported to be much less active in cells with Wnt

pathway activation. [ 39 ] Here, we hypothesized that LRP6 could be the main coreceptor

involved in the binding of a Wnt protein to its receptor (Frizzeld) and the coreceptors to

generate a β-catenin/TCF Wnt signal. In addition, DKK1 and WIF1 were the most significant

endogenous Wnt inhibitors comparing to SOST and Wnt3a involved in the reaction of

SJS/TEN in this study . Previous literature has also shown that DKK1 (eliciting inhibition of

LRP co-receptor function) and WIF1 (Wnt inhibitory factor 1) were both potential Wnt/β-

catenin signaling therapeutic targets for T cell responses and cytokines mediated

inflammatory autoimmune diseases. [ 40 ] Further study was needed to understand the

essential roles involved in the pathomechanism of SJS/TEN among these coreceptors and

endogenous Wnt inhibitors, and it may provide evidence for the potential biological targets

in the future.

The endogenous regulation in Wnt pathway in SJS/TEN. A previous study has

demonstrated the endogenous proteins/ligands (including LEF-1 and TCF-1) are

downregulated active Wnt signaling in effector T cells, which are the dominant immune cell

involved in SJS/TEN in our study. [ 23 ] These findings revealed consistent result between

endogenous and exogenous ligand during the activation of effector T cells. In addition, a

previous study had shown that endogenous LRP5 constitutively activates TCF/LEF-1 in the

absence of Wnt. [ 41 ] Another study also showed that removing the extracellular domain of

both LRP5 and LRP6 could lead to constitutive activation of the intracellular domain. In the

absence of exogenous Wnt 3a, full-length LRP6, but not LRP5, increased TCF/LEF-1

transcriptional activity, however both significantly potentiated Wnt 3a-induced TCF/LEF-1

activation. [ 42 ] Moreover, the intracellular domains (membrane-anchored and cytosolic) of

both LRP5 and LRP6 significantly increased TCF/LEF-1 activation in the absence of Wnt 3a,

and potentiated the Wnt 3a-induced decrease in beta-catenin phosphorylation, increase in

free beta-catenin levels and the increase in TCF/LEF-1 activity. [ 42 ] These findings have

demonstrated that LRP5 and LRP6 with and without the extracellular domain ( whether or

not they are membrane-anchored ) could still facilitate Wnt 3a-induced TCF/LEF-1 activation.

Taken together, the previous studies have shown the consistent results between exogenous

and endogenous regulation and both played important roles in modulating Wnt signaling.

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Active Wnt signaling also plays essential roles in the activation of other immune cells,

including antigen presenting cells and Tregs, which are also known to be central to the

pathomechansim of SJS/TEN.[3, 18, 43] The canonical Wnt pathway was involved in

suppressing dendritic cell activation and cross-priming of CD8+ T cell response.[40]

Activation of Wnt signaling could potentially increase the tolerogenicity of antigen

presenting cells. On the other hand, Tregs from the acute-stage TEN patients profoundly are

impaired in their suppressive function, resulting in more severe cytotoxic consequences.[44]

Interestingly, overexpression of stabilized beta-catenin in CD4+CD25+ Treg cells led to an

increase of the survival of these Treg cells cells and could further induce an anergic

phenotype in CD4+CD25- effector T cells, which favored the suppressive effect of Treg cells

[21]. In previous studies, Wnt signals may have the potential to restore Treg-mediated

suppressive actions, which may be beneficial to reverse the overwhelming cytotoxic T

lymphocyte mediated cytotoxicity.[45] However, the role of Wnt signaling in Tregs function is

still controversial since findings from another study showed that Wnt signals favored toward

Th17 instead of Tregs lineage.[46-48] It would be of interest and further investigations are

necessary to delineate the exact role of Wnt signaling in the regulation of Tregs in SJS/TEN.

SJS/TEN are lethal severe cutaneous adverse reactions. Although the incidence has

ameliorated by identifying the genetic predisposing factors, early diagnosis and prognosis

monitoring for SJS/TEN remain a challenge for clinical physicians. Besides, no acceptable

treatment guideline for this devastating condition highlights the inadequacy of the current

therapeutic remedies. Our data here, for the first time, reveal that Wnt signaling is

functionally involved in the immunopathogenesis of SJS/TEN. These findings suggest that

enhancing the activity of Wnt pathway may be a potential strategy to modulate immunity in

SJS/TEN and provide additional insight into therapeutic aspects of this devastating condition.

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Conflict of Interest

The authors declare that they have no relevant conflict of interest.

Acknowledgments

This study was supported by research grants from Ministry of Science and Technology,

Taiwan (MOST-108-2314-B-182A-006-MY3 to CB Chen, MOST-104-2314-B-182A-151-MY2 to

SC Su, and MOST-105-2628-B-010-007-MY3, MOST-106-2314-B-182A-037-MY3, to WH

Chung) and Chang Gung Memorial Hospital, Taiwan (CMRPG2H0081 and CMRPG2J0221 to

CB Chen; CIRPD1D0032, CORPG3F0041-3, OMRPG3E0041-3, and CORPG3F0061-3 to WH

Chung).

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Table 1. Demographic and clinical characteristics of SJS/TEN patients.

Demographic data SJS/TEN (n=25)

Age, years, mean SD (range) 52.5 18.7 (3-81)

Sex ratio (M:F) 9:16

Skin (TBSA, %, mean SD (range))

Erythema 56.5 26.7 (10–100)

Blister or detachment 30.9 22.9 (1–80)

Mucosa (n, (%))

Ocular 20 (80.0)

Oral 25 (100.0)

Genital 18 (72.0)

Systemic Complications (n, (%))

Hepatitis a 7 (28.0)

Acute kidney injury b 6 (24.0)

Gastrointestinal bleeding 6 (24.0)

Pneumonitis, pneumonia, or bronchiolitis

obliterans 4 (16.0)

Blood dyscrasia (n, (%))

Eosinophilia c 6 (24.0)

Leukocytosis d 4 (16.0)

Leukopenia e 9 (36.0)

Atypical lymphocytosis f 16 (64.0)

Thrombocytopeniag 7 (28.0)

Corneal ulcer or symblepharon (n, (%)) 5 (20.0)

Fever h (n, (%)) 13 (42.0)

Mortality (n, (%)) 5 (20.0)

Underlying disease, n

Malignancy 2

Chronic kidney disease 4

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Chronic liver disorder 3

Diabetes 3

Hypertension 9

Gouty 5

Epilepsy/Neuralgia 5

Rheumatoid arthritis 1

Culprit drugs, n

Anticonvulsants i 8

Antibiotics j 7

Allopurinol 4

Chlorzoxazone 1

Dapsone 1

NSAIDs 1

PPI 1

Sulfasalazine 2

SD, standard deviation; SJS, Stevens-Johnson syndrome; TEN, toxic epidermal necrolysis;

TBSA, total body surface area; NSAIDs, Non-Steroidal Anti-Inflammatory Drug; PPI, proton

pump inhibitor

a Values were 2 times greater than normal for glutamic-oxaloacetic transaminase, glutamic-

pyruvic transaminase, or total bilirubin.b The value of creatinine was 1.5 times greater than the normal value range (0.4–1.5 mg%)

after drug intake.c Eosinophils > 0.7 × 109/L, or > 5%, or >10% if leukocyte < 4.0 × 109/Ld The leukocyte count was greater than 11,000/μL.e The leukocyte count was less than 3,500/μL.f Abnormal lymphocytes present in blood.g The platelet count was lower than 150,000/μL.h Patients had a body temperature greater than 38.5°C.i Carbamazepine, lamotrigine, phenobarbital, and phenytoin

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i Cephalexin, flomocef, moxifloxacin, levofloxacin, and vancomycin1

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Figure Legends

Figure 1. Expression of Wnt signaling components is regulated in SJS/TEN and drug-

specific T cell activation. (A) Gene expression analyses of Wnt signaling components in

blister cells (BCs) and PBMCs of subjects with SJS/TEN. mRNA expression of LEF1, TCF1,

LRP5, and LRP6 was determined by real-time PCR. The respective mRNA levels were

compared among the blister cells (n=5) and PBMCs (n=5) from subjects with SJS/TEN at

active stage and control PBMCs from healthy donors (HD) (n=3). (B) Gene expression of Wnt

signaling components in drug-activated T cells from SJS/TEN patients. PBMCs obtained from

three SJS patients at active stage were cultured in RPMI1640 medium and stimulated

without (Control) and with the culprit drugs (MED), Solaxin, carbamazepine, and

vancomycin, respectively. After 7 days, the mRNA expression of LEF1, TCF1, LRP5, and LRP6

in drug-sensitive T cells, which were determined with the stimulation index (SI, calculated by

the level of secreted granulysin of stimulated to unstimulated cultures) greater than 2, was

measured by real-time PCR. *p <0.05; ** p <0.01; ***p <0.001; n.s, not significant; two-

sided Student’s t-test.

Figure 2. Intracellular LEF1 and TCF1 protein expression in CD8 T cell subsets of SJS/TEN

patients. TCF1 and LEF1 protein expression was detected in CD8 T cell subsets by

intracellular FACS staining. Black line histograms indicate TCF1 (A) or LEF1 (B) staining at the

active stage of disease course, and color line histograms represent staining at the recovered

stage. Representative flow cytometry plots show the expression levels of TCF1 (A) or LEF1

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(B) in peripheral CD8 T cells of SJS/TEN patients. A representative example from one SJS/TEN

patient is shown from four individual SJS/TEN patients. (C-D) The fraction of TCF1 high CD8 T

cells and LEF1 high CD8 T cells populations are increased at the recovery stage comparing with

that at the active stage by intracellular FACS staining though no statically significance

(p=0.18 and p=0.15, respectively).

Figure 3. Endogenous Wnt inhibitors associated with SJS/TEN. (A) Serum levels of DKK1,

SOST, WIF1, and Wnt3a were examined in 10 patients with SJS/TEN and 11 healthy donors

(HD) by ELISA. (B) The levels of DKK1, SOST, WIF1, and Wnt3a in blister fluid of 9 TEN

patients and 11 burn patients were measured by ELISA. * p< 0.05; ** p< 0.01; *** p< 0.001;

two-sided Student’s t-test.

Figure 4. IL-15 contributes to the attenuation of Wnt signaling in CD8+ T cells. (A) TCF1

protein expression was detected in CD8+ T cell subsets by intracellular FACS staining after

PBMCs were treated with cytokines indicated for 24 hr. Representative flow cytometry plots

were shown from one of three independent experiments. (B-E) We observed a significant

decrease in the portion of TCF1 high CD8 T cells in PBMCs from normal subjects that were

treated with IL-15, but not with IL-6, IL-8, or SDF-1 (stromal cell-derived factor-1, used as a

non-SJS-related cytokine) . * p < 0.05; two-sided Student’s t-test .

Figure 5. Drug-specific TCR engagement attenuates the Wnt signal transduction in

SJS/TEN. PBMCs of a carbamazepine (CBZ)-induced SJS/TEN patient were stimulated with

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the culprit drug (CBZ) or a tolerant drug (phenytoin, PHT) for 7 days. Levels of LEF1 and TCF1

protein expression were detected in CD8+ T cell subsets by intracellular FACS staining.

Representative flow cytometry plots were shown from one of five independent

experiments.

Figure 6. Activation of Wnt signaling attenuates activation of drug-specific T cells. PBMCs

of a carbamazepine (CBZ)- (A) or Ketoprofen-sensitive (B) SJS patient were obtained from

the heparinized blood by density centrifugation on Ficoll-Hypaque and stimulated with and

without the culprit drugs in the absence or presence of different concentrations of Wnt

signaling activator including LiCl (A) or SB216763 (B). After 7 days, the stimulation index (SI)

will be calculated by the level of secreted granulysin of stimulated to unstimulated cultures.

* p< 0.05; two sided Student’s t-test.

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Figure 1. Expression of Wnt signaling components is regulated in SJS/TEN and drug-

specific T cell activation.

(A) Gene expression analyses of Wnt signaling components in blister cells (BCs) and PBMCs

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of subjects with SJS/TEN. mRNA expression of LEF1, TCF1, LRP5, and LRP6 was determined

by real-time PCR. The respective mRNA levels were compared among the blister cells (n=5)

and PBMCs (n=5) from subjects with SJS/TEN and control PBMCs from healthy donors (HD)

(n=3). (B) Gene expression of Wnt signaling components in drug-activated T cells from

SJS/TEN patients. PBMCs obtained from three SJS/TEN patients were cultured in RPMI1640

medium and stimulated without (Control) and with the culprit drugs (MED), Solaxin,

carbamazepine, and vancomycin, respectively. After 7 days, the mRNA expression of LEF1,

TCF1, LRP5, and LRP6 in drug-sensitive T cells, which were determined with the stimulation

index (SI, calculated by the level of secreted granulysin of stimulated to unstimulated

cultures) greater than 2, was measured by real-time PCR. *p <0.05; ** p <0.01; ***p <0.001;

n.s, not significant; two-sided Student’s t-test.

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Figure 2. Intracellular LEF1 and TCF1 protein expression in CD8 T cell subsets of SJS/TEN

patients.

TCF1 and LEF1 protein expression was detected in CD8 T cell subsets by intracellular FACS

staining. Black line histograms indicate TCF1 (A) or LEF1 (B) staining at the active stage of

disease course, and color line histograms represent staining at the recovered stage.

Representative flow cytometry plots show the expression levels of TCF1 (A) or LEF1 (B) in

peripheral CD8 T cells of SJS/TEN patients. A representative example from one SJS/TEN

patient is shown from four individual SJS/TEN patients. (C-D) The fraction of TCF1 high CD8 T

cells and LEF1 high CD8 T cells populations are increased at the recovery stage comparing with

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that at the active stage by intracellular FACS staining though no statically significance

(p=0.18 and p=0.15, respectively).

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Figure 3. Endogenous Wnt inhibitors associated with SJS/TEN.

(A) Serum levels of DKK1, SOST, WIF1, and Wnt3a were examined in 10 patients with

SJS/TEN and 11 healthy donors (HD) by ELISA. (B) The levels of DKK1, SOST, WIF1, and

Wnt3a in blister fluid of 9 TEN patients and 11 burn patients were measured by ELISA. * p<

0.05; ** p< 0.01; *** p< 0.001; two-sided Student’s t-test.

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Figure 4. IL-15 contributes to the attenuation of Wnt signaling in CD8+ T cells.

(A) TCF1 protein expression was detected in CD8+ T cell subsets by intracellular FACS

staining after PBMCs were treated with cytokines indicated for 24 hr. Representative flow

cytometry plots were shown from one of three independent experiments. (B-E)

We observed a significant decrease in the portion of TCF1 high CD8 T cells in PBMCs from

normal subjects that were treated with IL-15, but not with IL-6, IL-8, or SDF-1 (stromal cell-

derived factor-1, used as a non-SJS-related cytokine) . * p < 0.05; two-sided Student’s t-test .

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Figure 5. Drug-specific TCR engagement attenuates the Wnt signal transduction in

SJS/TEN.

PBMCs of a carbamazepine (CBZ)-induced SJS/TEN patient were stimulated with the culprit

drug (CBZ) or a tolerant drug (phenytoin, PHT) for 7 days. Levels of LEF1 and TCF1 protein

expression were detected in CD8+ T cell subsets by intracellular FACS staining.

Representative flow cytometry plots were shown from one of five independent

experiments.

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Figure 6. Activation of Wnt signaling attenuates activation of drug-specific T cells.

PBMCs of a carbamazepine (CBZ)- (A) or Ketoprofen-sensitive (B) SJS patient were obtained

from the heparinized blood by density centrifugation on Ficoll-Hypaque and stimulated with

and without the culprit drugs in the absence or presence of different concentrations of Wnt

signaling activator including LiCl (A) or SB216763 (B). After 7 days, the stimulation index (SI)

will be calculated by the level of secreted granulysin of stimulated to unstimulated cultures.

* p< 0.05, two-sided Student’s t-test.

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